U.S. patent number 4,471,057 [Application Number 06/246,187] was granted by the patent office on 1984-09-11 for detection of colorectal carcinoma.
This patent grant is currently assigned to The Wistar Institute. Invention is credited to Meenhard Herlyn, Hilary Koprowski, Zenon Steplewski.
United States Patent |
4,471,057 |
Koprowski , et al. |
September 11, 1984 |
Detection of colorectal carcinoma
Abstract
Colorectal carcinoma is detected by testing body fluids for the
colorectal carcinoma monosialoganglioside identified by monoclonal
antibodies produced by fused cell hybrid ATCC HB 8059.
Inventors: |
Koprowski; Hilary (Wynnewood,
PA), Steplewski; Zenon (Strafford, PA), Herlyn;
Meenhard (Wynnewood, PA) |
Assignee: |
The Wistar Institute
(Philadelphia, PA)
|
Family
ID: |
22929644 |
Appl.
No.: |
06/246,187 |
Filed: |
June 30, 1981 |
Current U.S.
Class: |
436/518; 436/531;
436/542; 436/548; 436/804; 436/813 |
Current CPC
Class: |
C07K
16/3046 (20130101); G01N 33/57446 (20130101); Y10S
436/804 (20130101); Y10S 436/813 (20130101) |
Current International
Class: |
C07K
16/30 (20060101); C07K 16/18 (20060101); G01N
33/574 (20060101); G01N 033/54 (); G01N
033/56 () |
Field of
Search: |
;424/1,1.5,1.1,9,85,177
;23/23B,915,923
;436/548,536-542,504,518,531,53,543-545,547,548,804,813,815
;435/4,7,68,70,172,240,241,948 ;260/112R |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4172124 |
October 1979 |
Koprowski et al. |
4349528 |
September 1982 |
Koprowski et al. |
|
Other References
Herlyn et al., European Journal of Immunology, vol. 9, pp. 657-659,
(8-1979). .
Herlyn et al., Cancer Research, vol. 40, pp. 717-721, (3-1980).
.
Herlyn et al., International Journal of Cancer, vol. 27, pp.
769-774, (1981). .
Gilliland et al., Proc. National Academy Sciences, USA, vol. 77,
No. 8, pp. 4539-4543, (8-1980). .
Marx, J. L., Science, vol. 216, pp. 283-285, (4-1982). .
Herlyn et al., Proc. National Academy Sciences, USA, vol. 76, No.
3, pp. 1438-1442, (3-1979), "Colorectal Carcinoma". .
Koprowski et al., Aromatic Cell Genetics, vol. 5, pp. 957-972,
(1979). .
Koprowski et al., Science, vol. 212, pp. 53-55, (4-1981), "Specific
Antigen . . . ". .
Magnani et al., Science, vol. 212, pp. 55-56, (4-1981), "A
Monosialoganglioside . . . "..
|
Primary Examiner: Padgett; Benjamin R.
Assistant Examiner: Moskowitz; M.
Attorney, Agent or Firm: Banner, Birch, McKie &
Beckett
Government Interests
The invention described herein was made in the course of work under
a grant or award from the Department of Health, Education and
Welfare.
Claims
We claim:
1. A method for the detection of colorectal carcinoma which
comprises providing body fluid from an individual and testing said
body fluid for the presence of the colorectal carcinoma
monosialoganglioside identified by the antibody produced by the
fused cell hybrid ATCC HB 8059.
2. The method of claim 1 wherein the body fluid is serum.
3. An assay for the detection of colorectal carcinoma which
comprises testing body fluid for the presence of the colorectal
carcinoma monosialoganglioside identified by the antibody produced
by the fused cell hybrid ATCC HB 8059, hereafter referred to as KS
monosialoganglioside, by the procedure comprising:
(a) incubating an aliquote of anti-KS monosialoganglioside
monoclonal antibodies produced by a fused cell hybrid with an
aliquot of cell free body fluid from an individual;
(b) incubating the mixture from (a) above in contact with a surface
having said KS monosialoganglioside attached thereto;
(c) washing said mixture from said surface after said incubation of
(b);
(d) measuring the amount of antibody bound to said surface; and
(e) comparing the results obtained with results obtained when steps
(a) to (d) are performed but a buffer solution is employed in step
(a) to replace the body fluid.
4. The assay of claim 3 wherein the body fluid is serum.
5. The assay of claim 3 or 4 wherein a radioimmunoassay is employed
to measure the antibody bound to said surface.
6. The assay of claim 3 or 4 wherein assay steps (a) through (d)
are also performed employing a nonspecific innunoglobulin for the
species of animal from which the antibody-producing cell of the
fused cell hybrid was obtained instead of the serum in step
(a).
7. The assay of claim 3 or 4 wherein the antibody is produced by
fused cell hybrid ATCC HB 8059.
8. The assay of claims 3 or 4 wherein the antibody employed
interferes with the binding of the antibody produced by fused cell
hybrid ATCC HB 8059.
Description
DESCRIPTION OF THE INVENTION
Most of the tests developed for immunodiagonosis of digestive
system tumors by detection of circulating tumor-associated antigens
such as carcinoembryonic antigen (CEA), alpha-fetoprotein and
others lack the specificity to make a confident distinction between
cancer and control groups of patients. Particularly disturbing is
the fact that many healthy persons or patients with a non-malignant
disease circulate within their bodies of one or another of the
antigens purported to be of specific diagnostic value for cancer
patients. For example, extensive exploration of carcinoembryonic
antigen (CEA) as a diagnostic tool for colorectal cancer (CRC)
revealed that, although most CRC patients have CEA in their sera,
many sera obtained from patients with ulcerative colitis, alcoholic
cirrhosis, pulmonary emphysema and other primary site carcinomas,
also bind anti-CEA antibody in radioimmune assays (RIA).
Furthermore, about one fifth of all healthy subjects who smoke
cigarettes also have CEA in their sera.
There is an obvious need for an assay that will identify, with a
good degree of reliability, those individuals who have colorectal
carinoma and which will not provide a spectrum of disturbing and
expensive false positives when healthy patients are tested.
It is one object of this invention to provide a test that has
substantial specificity for colorectal carcinoma.
It is another object of this invention to provide a hybrid cell
which produces an antibody that has substantial specificity for
colorectal carcinoma.
According to this invention there is provided a method for the
detection of colorectal carcinoma which comprises testing body
fluid for the presence of the colorectal carcinoma
monosialoganglioside identified by the antibody produced by the
fused cell hybrid ATCC No. HB 8059.
In one specific aspect of this invention there is provided an assay
for the detection of colorectal carcinoma which comprises testing
body fluid for the presence of the colorectal carcinoma
monosialoganglioside identified by the antibody produced by the
fused cell hybrid ATCC No. HB 8059, hereafter referred to as KS
monosialoganglioside by the procedure comprising:
(a) incubating an aliquot of anti-KS monosialoganglioside
monoclonal antibodies produced by a fused cell hybrid with an
aliquot of cell free body fluid from an individual;
(b) incubating the mixture from (a) above in contact with a surface
having said colorectal carcinoma monosialogangliosides attached
thereto;
(c) washing said mixture from said surface after said incubation of
(b);
(d) measuring the amount of antibody bound to said surface; and
(e) comparing the results obtained with results obtained when steps
(a) to (d) are performed but a buffer solution is employed in step
(a) to replace the body fluid.
This invention permits the detection of colorectal carcinoma not
only in clinically diagnosed CRC patients but also in patients in
an early stage of disease, when prompt surgical intervention may be
warranted.
In accordance with this invention, it has been discovered that an
extra-cellular antigen is present in the body fluid of CRC patients
that cannot be detected in healthy subjects, in patients suffering
from chronic inflammatory disease of the bowel or in patients
suffering from other cancers. The only exception to this was
detection of the antigen in two cases of pancreatic carcinoma and
two cases of gastric carcinoma, carcinomas which have the same
endodermal origin as CRC cells. Presence of the antigen in this
case may be advantageous, given the usual difficulty in diagnosing
this type of cancer during its early growth stages.
The monoclonal antibodies contemplated for use in this invention
bind specifically to an antigen of CRC cells and not to an antigen
of cells of other cancers, with the exception of carcinomas of the
stomach and of the pancreas maintained in culture, which tumors
have the same endodermal origin as CRC cells. The nature of the
antigen recognized by the monoclonal antibody may account for the
high specificity of reaction. It is not CEA, since binding of
anti-CRC antibody to CRC cells was not inhibited by purified CEA
obtained from the diagnostic kit of either Hoffman La Roche or
Abbot Diagnostics. Moreover, no correlation was observed between
the presence of CEA in the blood of patients and healthy volunteers
and the results of the inhibition assay of this invention. Relation
of the antigen detected by the anti-CRC antibody to other
tumor-associated antigens such as B.sub.2 -microglobulin can be
ruled out, since the antigen recognized by anti-CRC antibody is not
a protein: the antigen is stable under conditions such as
incubation at 37.degree. C. for two weeks, drying and heating to
85.degree. C. Studies have indicated that the antigen is a
monosialoganglioside not found in normal adult tissues.
The KS-monosialoganglioside antigen is present in the lipid extract
of CRC cells and is identified by the antibody produced by fused
cell hybrid ATCC No. HB 8059, deposited with the American Type
Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md.
20852. A variety of known testing procedures can be employed to
test for its presence in body fluids, such as blood, cell free
serum or urine. Such procedures include without limitation,
radioimmunoassay with radio labled monoclonal antibodies, an
agglutination inhibition assay, an enzyme linked assay, chemical
analysis, and the like. Such assays can be carried out with the
fluids containing such cells as may be present but preferably are
carried out with cell-free fluids. The inhibition assay employing
monoclonal antibodies is a preferred assay for the practice of this
invention.
Antibodies useful in this invention can be obtained by challenging
an animal with a monosialoganglioside-containing CRC cell fraction
from a patient with colorectal carcinoma, forming a fused cell
hybrid between antibody producting cells from said animal and
myeloma cells, cloning said hybrids and selecting clones which
produce antibodies that bind with said KS monosialoganglioside.
Techniques for challenging animals, preparing fused cell hybrids
and testing for the binding capacity of antibodies are known to the
art and are described, inter alia, in U.S. Pat. No. 4,172,124,
Somatic Cell Genetics, Vol. 5, No. 6, pp 957-972 (1979), and
Kennett, Monoclonal Antibodies, Plenum Press (1980). The particular
species of animal from which the myeloma and antibody producing
cells are selected are not critical insofar as it is possible to
use cells of the same species or to fuse cells of one species with
another, for example, mouse to mouse, rat to rat or mouse to
rat.
Antibodies which interfere with the binding of antibodies produced
by hybrid cell line ATCC No. HB 8059 are preferred for the assay of
this invention. Antibodies corresponding to those produced by
hybrid cell line ATTC No. HB 8059 are particularly preferred for
the assay of this invention.
The inhibition assay can employ an aliquot of a cell free body
fluid including serum or urine. Blood serum obtained by removing
the cells from blood is preferred and, for ease of presentation,
the following description will be in terms of serum. It is to be
understood, however, that other body fluids are expressly
contemplated for use in this assay.
According to the assay, an aliquot of serum is incubated with an
aliquot of anti-KS monosialoganglioside antibody (i.e., the
antibody is permitted to bind to appropriate sites in the serum if
such sites are present). After the initial incubation period, the
mixture is brought into contact with a surface having attached
thereto a monosialoganglioside antigen from a known human CRC cell
and again allowed to incubate. For many of the tests herein
reported, the human CRC cell line used was SW 1116 obtained from
Scott and White Clinic, Temple, Tex. and described, inter alia, in
Somatic Cell Genetics, Vol 5, No 6, 1979 pp 957-972. The choice of
a particular CRC cell line with its attendant KS
monosialoganglioside is not critical and many such cells are
readily available to the art.
After the second incubation, the mixture is separated from the
surface (e.g., by washing) and the amount of antibody bound to the
surface is measured. Such measurement can be accomplished using
techniques known to the art including, for example, an enzyme
linked assay, immuno fluorescences, and radioimmunoassay. Typical
techniques are described, inter alia, in Kennett, Monoclonal
Antibodies, Plenum Press, 1980.
For a radioimmunoassay, the radioactive labeled immunoglobulin
should be for the animal species from which the antibody-producing
cell of the fused cell hybrid was obtained. The assays described
herein employed mouse-mouse fused cell hybrids and [.sup.125 I]
rabbit anti-mouse immunoglobulin. In such assay, the surface,
following removal of the serum-antibody mixture, was incubated with
the radio-labeled anti-mouse immunoglobulin so that it could bind
to any monoclonal antibodies that had bound to the surface in the
previous incubation.
The precoating of surfaces to provide a surface having an antigen
attached thereto is a technique that is known in the art. Such
techniques, for example, may use commercially available polyvinyl
plates or beads. Indeed, if desired, CRC cells which contain the
antigen on their surface can be employed. The primary requisite is
a solid to which the monoclonal antibody can bind and thereafter be
separated from the liquid mixture.
Maximum binding of antibodies may be determined by incubation of
the antibodies with an aliquot of buffer solution instead of the
serum. Appropriate buffer solutions for such use are well known in
the art and include, inter alia, a phosphate buffered saline
solution containing 1% gamma globulin-free horse serum. The
antibody-buffer mixture is then incubated with the antigen
containing surface and the amount of antibodies which bind to the
surface are measured as discussed above.
Background controls may be determined by the incubation of test
samples containing serum with aliquots of nonspecific
immunoglobulin for the animal species from which the
antibody-producing cell of the fused cell hybrid was obtained.
While use of background controls is described below and provides
best accuracy, the use of such controls is not essential to an
effective assay. Direct comparison of test assays with maximum
binding assays can also be employed. If background controls are
employed the percent inhibition may be calculated according to the
formula described below.
The inhibition assay utilizes the competitive binding of an
antigen. If the patient's serum contains the antigen from a colon
cancer, it will react with the monoclonal antibody forming a bound
antibody antigen complex. This complex is no longer able to react
in another binding reaction since it has already been bound. If
there is no carcinoma antigen in the patients's serum, the
monoclonal antibody is not bound and is still reactive.
The mixture is then transferred into contact with a surface having
attached KS monosialoganglioside antigen to (e.g., a second small
culture well in which has been placed a preparation of human colon
cancer cell extract). These have a known amount of the antigen. If
in the myriad of material transferred to this well there is free or
unbound antibodies this will react with the cell antigen. The
amount of antibody can be detected by binding a radioactive isotope
to the mouse antibody and the amount of antibody present bound to
cells is detected by the amount of isotopes present. In this test
the more bound isotope that is present in the second well, the less
colon tumor antigen that was present in the first culture, on the
other hand, if there is little bound isotope in the second well,
the rest was present in the patient's serum. If there was none
present in the patient's serum, then he is judged not to have
carcinoma, but more importantly if there is material detected in
his serum this test has confirmed that he does have colon
carcinoma.
In order to illustrate the inhibition assay of this invention more
fully, the following is presented as a represntative procedure
employing antibodies produced by a mouse-to-mouse fused cell hybrid
which utilized P3.times.63 Ag8 mouse myeloma cells.
Hybridomas secreting anti-CRC antibody into the medium were grown
according to the method described in Somatic Cell Genetics, Vol 5,
No 6, pp 957-972 (1979). The hybridoma medium contained
approximately 10 .mu.g antibody per ml. Indirect radioimmunoassay
(RIA) for antibody binding to its target is also described in the
Somatic Cell Genetics article above. Dilutions of hybridoma
antibodies were used that exhibited 40 to 50 percent maximal
reactivity against a given concentration of target antigen prepared
either as membrane extracts or as serum-free tissue culture media
(SFTCM) of cell line SW 1116. Diluted antibody to be tested for
inhibition was mixed in gelatin-coated polyvinylchloride plates
(Cooke Eng. Co., Alexandria, Va.) with equal volume of dilutions of
human sera. The mixtures were incubated overnight in a humidified
chamber at 4.degree. C. and then transferred to wells of plates
precoated with either membrane extracts or serum-free supernatant
of SW 1116 cells. After overnight incubation, the plates were
washed with cold Vernoal-buffered NaCl solution and then again
incubated overnight at 4.degree. C., this time with [.sup.125 I]
rabbit IgG anti-mouse F(ab).sub.2. The plates were then washed and
the wells separated and counted in a Packard gamma counter.
The percentage of specific inhibition of binding of hybridoma
antibodies was calculated from the mean of triplicate wells
according to the formula: ##EQU1##
Maximum binding was determined through the incubation of hybridoma
antibodies with buffer solution instead of serum samples, and
background controls were determined through the incubation of test
samples with nonspecific mouse immunglobulin (supernatant from
myeloma P3.times.63 Ag8). Statistically significant differences in
inhibitions of binding between the test and control samples can be
calculated for each test by the Student's t-test.
The following examples are included for illustrative purposes only
and are not intended to limit the scope of the invention.
EXAMPLE 1
BALB/c mice were challenged with a monosialoganglioside of
colorectal carcinoma cells (by injection with immunizing colorectal
carcinoma cells) and a fused hybrid was obtained from the mouse
spleen cells and mouse myeloma cell line P3.times.63 Ag8. The above
procedures were carried out as described in Somatic Cell Genetics,
Vol 5, No 6, pp 957-972 (1979). Antibody producing fused cell
hybrid, ATCC HB 8059 (Wistar No. SW-1116-19-9) was selected and its
antibodies were employed in the assays herein.
EXAMPLE 2
Sera were obtained from 33 patients who had advanced adenocarcinoma
of the colon and rectum with documented metastases in their liver
or lungs, 6 patients with other bowel diseases, including two with
multiple polyps and 38 patients with forms of cancer other than
colorectal carcinoma, all hospitalized at the Onocologic Hospital,
Fox Chase Medical Center, Philadelphia, Pa. The sera were
aliquoted, coded and frozen at -70.degree. C. Preoperative patients
and those with suspected but unproven recurrent cancer were not
included in this study group. An additional 13 sera obtained from
CRC patients were provided by Met Path Inc., Bala Cynwyd, Pa. Sera
were also obtained from 39 healthy volunteers: 15 females 20 to 40
years of age, ten females 41 to 65 years of age; nine males 20 to
40 years of age and five males 41 to 65 years of age. Fifteen of
these 39 healthy subjects were smokers.
Cells of CRC SW-1116 were used as a source of target antigen in the
binding assay. This tumor is maintained in tissue culture and
secretes its antigen in the tissue culture medium. Either extracts
(3M KCl) of the cell membranes or cell-free tissue culture medium
(SFTCM) were used.
Assays were conducted as described above. In this assay, a 1:500
dilution of anti-CRC monoclonal antibody was mixed with a dilution
of either 1:3 or 1:9 of patients' sera and, after incubation,
transferred to wells of plates precoated with 3M KCl membrane
extract of SW 1116 tumor cells containing 7 .mu.g protein per well.
Inhibition of binding was then determined by RIA. The results are
shown in Table 1.
TABLE 1 ______________________________________ NUMBER OF PATIENTS
CLINICALLY % Inhibition DIAGNOSED AS: by Human Sera Advanced Other
Other of Binding Colorectal Bowel Malig- Healthy of Antibody
Carcinoma Diseases nancies Volunteers
______________________________________ <10% 3 (10) 5 (98) 34
(90) 39 (100) 10-19% 6 (18) 1 (2) 2 (5) 0 over 19% 24 (72) 0 2 (5)
0 TOTALS: 33 6 38 39 ______________________________________ () =
refers to percentage of total number of patients.
Of 33 sera from patients with advanced CRC, 24 (72 percent)
inhibited binding of anti-CRC hybridoma antibody more than 19
percent (p<0.025). Six sera inhibited 10 to 19 percent, and
three sera inhibited binding by less than 10 percent. In an
additional study of the three patients whose sera inhibited less
than 10 percent of the binding reaction, ascitic fluid obtained
from one of the patients inhibited binding by 30 percent. This and
another patient in the 0 to 10 percent inhibition group showed
greater than 20 percent inhibition in serum samples collected at
different times.
The above results compared strikingly with the lack of inhibition
obtained with 39 sera from health volunteers of different ages,
some of whom were heavy smokers; sera of this group inhibited
binding an average of 2.6 percent. Of 38 sera obtained from
patients with malignancies including 6 breast carcinomas and 17
melanomas, none inhibited binding significantly. The only
exceptions to this finding were sera obtained from two patients
with carcinoma of the pancreas, which sera inhibited binding by 35
percent and 60 percent and from 2 patients with gastric carcinomas
(10-19% range). Sera obtained from three patients with inflammatory
bowel diseases (one with colitis, one with proctitis and one with
diverticulitis), or from two patients with multiple colonic polyps
did not inhibit binding. Serum from one patent, diagnosed as having
colitis, inhibited binding in the 10-19% range. It is too soon to
exclude the presence of a malignancy in that patient. Each serum
listed in Table 1 was tested as a coded sample at least three times
in the inhibition assay with reproductible results.
Serum was used freshly draw or frozen and thawed once; sera that
have been frozen and thawed several times give erratic results.
EXAMPLE 3
The assay of Example 2 was repeated except that (CRC) SW 1116 SFTCM
or cell membrane extract from SW 1116 tumor cells was employed
instead of sera. As a control, the same material obtained from WM
47 human melanoma was used to inhibit the binding reaction. The CRC
cell membrane extract at concentrations of 5 to 20 g protein per
well and SFTCM at concentrations of 10 to 20 .mu.g protein per well
inhibited binding of the anti-CRC monoclonal antibody to the target
antigen preparation. No inhibition was observed with melanoma SFTCM
or melanoma cell membrane extract.
EXAMPLE 4
The following tests were conducted in order to identify the nature
of the antigen.
Total lipid extracts of colorectal carcinoma and melanoma cells,
and of meconium were obtained according to a method devised for the
quantitative extraction of gangliosides from brain tissue and
described in Biochem Biophys Acta, 617:97-109. One gram wet weight
of tissue culture cells or meconium was homogenized in 3 ml H.sub.2
O at 4.degree. C. with a Potter-Elevhjem homogenizer. The
homogenate, followed by 5.4 ml chloroform, was added to 10.8 ml
methanol with constant stirring. After stirring at room temperature
for 30 minutes, the extract was centrifuged at 15,000.times.g for
10 minutes. The pellet was rehomogenized in 2 ml H.sub.2 O and
extracted as above with 8 ml chloroform-methanol (1:2). The
supernatant solutions from both extractions were combined and
evaporated under a stream of dry nitrogen, and the residue was
either dissolved in chloroform-methanol (2:1) for application to
thin layer chromatography sheets or dissolved in methanol for
DEAE-Sephadex chromatography as described in Ledun, Research
Methods in Neurochemistry, Plenum Pub. Co., 1978, 371-410.
The antigen was detected on thin layer chromatograms by
autoradiography with the following modification of a method
developed for the detection of gangliosides that bind to cholera
toxin: thin layer chromatography sheets (chromagram 100 m thick,
10.times.10 cm, Eastman Kodak Co., Rochester, N.Y.) were scribed to
create twenty 0.5 cm.times.10 cm lanes. Samples of 1 .mu.l of total
lipid extract suitably diluted were spotted on the lanes 1.5 cm
from the bottom. The sheet, clamped in a sandwich chamber, was
developed in a chromatography tank containing
chloroform-methanol-0.25 percent KCl (60:35:8), air-dried, and then
soaked for 10 minutes at 4.degree. C. in 0.01M sodium phosphate
buffer, pH 7.2, containing 0.15M NaCl, 1 percent
polyvinylpyrrolidone (M.sub.r 40,000 pharmaceutical grade; Sigma
Chemical Co., St. Louis, Mo.), and 0.1 percent sodium azide (Buffer
A). The wet chromatogram was laid horizontally on a slightly
smaller, parafilm-covered, glass plate. Serum-free hybridoma
culture medium containing about 10 .mu.g antibody per ml.sup.1 was
diluted 1:4 with Buffer A and gently pipetted onto the chromatogram
(about 50 .mu.l/cm.sup.2 of chromatogram). After incubation in a
humid atmosphere for 6 hours at 4.degree. C., the chromatogram was
washed by dipping in six successive changes of 0.01M sodium
phosphate buffer, pH 7.2, containing 0.15M NaCl (Buffer B). The
chromatogram was then laid horizontally as before and immediately
layered with [.sup.125 I] rabbit IgG antimouse F(ab).sub.2 in
Buffer A (10.sup.6 cpm/ml; about 50 .mu.l/cm.sup.2 of
chromatogram). After incubation in a humid atmosphere for 12 hours
at 4.degree. C., the chromatogram was washed six times in Buffer B,
air-dried and exposed to XR-2 X-ray film (Eastman Kodak, Col,
Rochester, N.Y.) for 50 hours.
Colorectal carcinoma cells were treated with ficin (Sigma Chemical
Co., St. Louis, Mo., 2.times.crystallized) and with Vibrio cholerae
neuraminidase (Calbiochem, La Jolla, Ca.) as described in Proc.
Natl. Acad. Sci. USA, 1977, 74:4591-4594.
The binding of the hybridoma antibody to colorectal carcinoma cell
line SW 1116 was unaffected by pretreatment of the cells with ficin
but abolished by pretreatment of the cells with neuraminidase. This
behavior is consistant with the antigen being a ganglioside. Total
lipid extracts of cells, which contain gangliosides, were
chromatographed and tested for antigen by autoradiography. Antigen
was detected in extracts of colorectal carcinoma cells but not in
extracts of human melanoma cells, which do not bind antibody. The
antigen migrates between the standard gangliosides G.sub.M1 and
G.sub.D1a under the conditions employed.
Upon DEAE-Sephadex chromatography of the total lipid extract, the
antigen was bound to the DEAS-Sephadex and was eluted from the
resin in the monosialoganglioside fraction 0.04M Ammonium acetate
in methanol. No antigen was detected either in the
disialoganglioside fraction eluted by 0.12M ammonium acetate in
methanol or in the tri- and tetrasialoganglioside fraction eluted
by 0.5M ammonium acetate in methanol. This property and the
antigen's chromatographic mobility is indicative of a large
monosialoganglioside.
The antigen was not detected by the autoradiographic method in
ganglioside mixtures from human tissues other than colorectal
carcinoma. However, the antigen was present in human meconium,
which is a rich source of fetal glycolipids.
Since modifications will be apparent to those skilled in the art,
it is intended that this invention be limited only by the scope of
the appended claims.
* * * * *